To date, 51 human adenovirus serotypes have been identified that are subdivided into 6 subgroups (A, B, C, D, E and F) based on hemagglutination properties and sequence homology (Francki et al. 1991). The adenovirus infectious cycle is divided into an early and a late phase. In the early phase, the virus is uncoated and the genome is transported to the nucleus, after which the early gene regions (E1, E2, E3 and E4) become transcriptionally active. The E1 region contains two transcription regions: E1A and E1B. E1A encodes proteins that are involved in modification of the host cell cycle and activation of the other viral transcription regions (reviewed by Russell 2000). The E1B region encodes two major proteins, E1B-19K and E1B-55K, that prevent the induction of apoptosis resulting from the activity of the E1A proteins (Rao et al. 1992; Yew and Berk 1992; Shenk 1996). In addition, the E1B-55K protein is required in the late phase for selective viral mRNA transport and inhibition of host protein expression (Pilder et al. 1986). E2 is also divided in two subdomains, E2A and E2B, that together encode three proteins (a DNA-binding protein, a viral polymerase and a pre-terminal protein) that are all involved in replication of the viral genome (Van der Vliet 1995). E3 is not necessary for replication in vitro but encodes several proteins that subvert the host defense mechanism towards viral infection (Horwitz 2001). E4 harbors at least six open reading frames (orfs) that encode proteins involved in several distinct functions related to viral mRNA splicing and transport, host cell mRNA transport, viral and cellular transcription and transformation (reviewed by Leppard 1997). The late proteins, necessary for formation of the viral capsids and packaging of viral genomes, are all generated from the major late transcription unit (MLTU) that becomes fully active after the onset of replication. A complex process of differential splicing and polyadenylation gives rise to more than 15 mRNA species that share a tripartite leader sequence. The E1B-55K, E4-orf3 and E4-orf6 proteins play a pivotal role in the regulation of late viral mRNA processing and transport from the nucleus. For this process E1B-55K interacts with E4-orf6 to form a functional complex that stimulates transport of viral mRNAs to the cytoplasm, while the complex is also involved in inhibition of the transport of cellular mRNAs from the nucleus to the cytoplasm (reviewed in Leppard 1997 and 1998).
Production of E1-deleted vectors based on subgroup C serotypes Adenovirus serotype 5 (Ad5) or Adenovirus serotype 2 (Ad2) is achieved in E1-complementing cell lines, such as 293 (Graham et al. 1970), 911 (Fallaux et al. 1996) and PER.C6™ (Fallaux et al. 1998; ECACC deposit no. 96022940). As disclosed in WO 99/55132 and WO 01/05945, vectors and cell lines can be matched to avoid generation of replication-competent adenoviruses through homologous recombination between adenovirus sequences in the cell line and the vector. For efficient production of replication-incompetent adenoviruses derived from group C, the cell line PER.C6™ is preferably used. Using this cell line, adenovirus vectors can be matched, thereby allowing for producing group C adenoviral vectors in the absence of replication-competent adenovirus (Fallaux et al. 1998; U.S. Pat. No. 6,033,908). However, group C vectors may not always be the ideal vehicles for direct in vivo applications since the infection efficiency is seriously hampered by the presence of high titers of neutralizing activity in most humans and the absence of sufficient amounts of the cellular receptor (Coxsackie-adenovirus receptor, CAR) on specific primary target cells (e.g., endothelial cells, smooth muscle cells, synoviocytes, monocytes and dendritic cells). Administration of higher amounts of viruses to increase transduction may lead to increased toxicity and unpredictable clinical outcome due to the variation in neutralizing titers of subjects that are treated. These limitations can be overcome by the use of other serotypes of adenoviruses. For example, in the receptor-binding part of a fiber of subgroup B viruses (in particular of serotype 16), when expressed on an Ad5-based vector, Ad5-based vector-mediated infection is significantly increased in human endothelial cells and smooth muscle cells (WO 00/31285) and in human synoviocytes (WO 00/52186). The fiber of another subgroup B adenovirus, Ad35, is most efficient in mediating infection of human monocytes and dendritic cells (WO 00/03029). Furthermore, Ad35 has been identified as a virus to which the vast majority of the human population has no neutralizing activity (WO 00/70071).
There is a generally felt need in the art to develop technology that has broader serotype utility. A particular problem is the lack of suitable packaging cell lines for these other serotypes. Packaging cell lines for Ad5 vectors typically comprise E1-encoded proteins derived from adenovirus serotype 5. Examples of such “standard” packaging cell lines are 293, 911 and PER.C6™. Attempts to produce vectors derived from other serotypes on these standard packaging cell lines have proven arduous, if not unsuccessful. Occasionally, some production is seen, depending on the particular serotype used. However, yields are poor from recombinant adenovirus vectors derived from adenovirus subgroups other than subgroup C and produced on cell lines transformed and immortalized by E1 from Ad5. In a paper by Abrahamsen et al. (1997), improved plaque purification of an E1A-deleted adenovirus serotype 7 vector (subgroup B) was observed on 293 cells comprising E4-orf6 derived from adenovirus serotype 5, as compared to 293 cells lacking the E4-orf6 sequence from Ad5. However, a problem was encountered with the stability of the vector as unexpected recombinations were observed in plaque-purified stocks. An additional problem was encountered with wild-type adenovirus virus contamination during production. Moreover, for large-scale production of adenoviruses, it is not useful to co-transfect E4-orf6 to obtain titers that are high enough for application. One option for growing such adenoviruses is to provide cells with the E4-orf6 gene stably integrated into the genome of the complementing/packaging cell line. Such cells have been described in the art (e.g., WO 96/22378). A disadvantage of that system is the fact that new stable cell lines have to be generated and numerous selection rounds have to be performed before stable and proper cells have been generated. This process is laborious and time consuming. In general, it can be stated that generation and propagation of adenoviruses from serotypes other than serotype 5 (subgroup C), such as subgroup B viruses, has proven to be difficult on Ad5-complementing cells. As has been disclosed in WO 00/70071, recombinant viruses based on subgroup B virus Ad35 can be made by co-transfection of an expression construct containing the Ad35 early region-1 sequences (Ad35-E1). Furthermore, Ad35-based viruses that are deleted only for E1A sequences and not for E1B were shown to replicate efficiently on PER.C6 cells, suggesting that the E1A proteins of Ad5 are able to complement the Ad35-E1A functions (applicant's international application PCT/NL01/00824, not yet published). Moreover, the experiments show that lack of Ad35-E1B results in poor yields on Ad5-complementing cells. WO 00/70071 also discloses cell lines for producing E1-deleted non-group C adenoviral vectors by further modifying cell lines that are capable of complementing adenovirus serotype 5. WO 00/70071 further suggests that one should establish new cell lines harboring Ad35-E1 sequences for the complementation of recombinant adenovirus serotype 35 vectors lacking the E1 region (see also applicant's international application PCT/NL01/00824). However, as also discussed above, if one desires to apply a specific serotype for a specific need, one would have to establish a new cell line for every specific serotype or modify the available cell lines that can complement adenovirus serotype 5 for complementation of the serotype of interest. It would clearly be advantageous to use the established cell lines that are available in the art and not to modify these and use them for producing all other, non-Ad5 serotypes, applying the established and efficient methods known in the art. It is concluded that until the invention, no flexible and proper “production platform” was available in the art that enabled one to produce useful yields of adenovirus serotypes that were different from the serotypes of subgroup C.